Page 192 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

2. Label target cells with IncuCyte pHrodo Orange Cell 3. Add treatment to effector cells
Labeling Kit 3.1 During target cell incubation, use a multichannel pipette
NOTE: Grow enough target cells in advance to to transfer 25 μL from the treatment plate containing
accommodate the different cell densities required to set the antibody, isotype control, and vehicle control to the
up the assay. We recommend testing target-to-effector effector cell plate. Incubate for 15-30 minutes at 37 °C.
cell ratios of 20:1, 10:1 and 5:1.
2.1 Harvest target cells and transfer into a 50 mL centrifuge 4. Add IncuCyte® pHrodo® labeled target cells to effector cells
tube. Centrifuge for 7 minutes at 1000 rpm. 4.1 Create two-fold serial dilutions of the labeled target cells
2.2 Aspirate supernatant and resuspend cell pellet with 50 by creating a 7-point, two-fold serial dilution (500,000
mL IncuCyte pHrodo Wash Buffer (component cells/well to 7,812 cells/well).
C). Gently mix cells by trituration and determine cell
count using a hemocytometer.
2.3 Harvest cells by centrifugation for 7 minutes at 1000 Target Cell Cell no. Volume
rpm. Aspirate IncuCyte pHrodo Wash Buffer and resuspend Seeding Density Per Well Per Well
cell pellet in IncuCyte pHrodo Labeling Buffer (component
D) to a density of 1x106 cells/mL. 1x10 cells/mL 250,000 25 μL
7
2.4 Reconstitute IncuCyte pHrodo Orange Cell Labeling Dye 5x10 cells/mL 125,000 25 μL
6
(component A) in 100 μL of DMSO (component B) to create 6
a stock concentration of 1 mg/mL. 2.5x10 cells/mL 62,500 25 μL
2.5 Add the solubilized IncuCyte pHrodo Orange Cell
Labeling Dye to the target cell suspension at the
concentration determined during optimization (refer 4.2 Immediately following target cell resuspension, remove
to Target Cell Labeling Optimization under General the effector cell plate from the incubator and add the
Guidelines). Incubate the centrifuge tube containing target cell suspensions to the cell plate (25 μL per well)
cells for 1 h at 37 °C. using a multichannel pipette.
2.6 Remove excess IncuCyte pHrodo Orange Labeling Dye 4.3 Remove bubbles and immediately place plate in the
from cells: IncuCyte S3 for Neuroscience (refer to Data Acquisition and
a. Centrifuge the cell:labeling dye suspension at 1000 Analysis section).
rpm for 7 minutes. Aspirate off supernatant and
resuspend apoptotic target cells in 50 mL of target cell
media.
b. Harvest cells by centrifugation for 7 minutes at 1000
rpm. Aspirate supernatant and resuspend apoptotic
target cells in effector cell media to yield a cell density
7
of 1 x 10 cells/mL.

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